Myosin IX is a member of the diverse myosin superfamily and distributed in a variety of tissues, however, its physiological function is unclear. A most intriguing finding is that myosin IX is a single headed processive motor with reverse directionality. Furthermore, it contains Rho GAP domain in its tail and Ras binding domain in its head domain, suggesting its function as a motor protein carrying signaling function. However, myosin IX function and regulation at a molecular level are largely unknown. The goal of the proposed project is to clarify the molecular mechanism of function and regulation of myosin IX. First, we will study how the single-headed myosin IX can move processively on actin filaments. The best approach to show the processive movement of myosin IX is the use of single molecule analysis. We will employ two techniques, i.e., mechanical measurement with optical trap nanometry and direct visualization of the movement by total internal reflection (TIRF) microscopy. The rotational motion of myosin IX on actin will be monitored by visualizing the movement of bead attached myosin IX on actin filament. Second, the conformational changes of myosin IX during the mechanical cycle will be studied by single molecule polarization TIRF microscopy that measures the angular change of myosin head, X-ray solution scattering that can determine the overall structural changes of myosin IX with 0.1nm resolution, and 3D image reconstitution of the structure of actin filament decorated with myosin IX with cryo-electron microscopy. Third, the kinetic analysis of acto-myosin IX will be done. The cross-bridge cycling of myosin is closely related with the myosin's ATPase cycle, therefore, the analysis of each kinetic step of acto-myosin IX is anticipated to yield the critical information to understand the motor mechanism of myosin IX. Fourth, we will clarify the structural elements that determine the processivity and the reverse directionality of myosin IX. Our recent finding that Rho kinase activates myosin IX motor activity sheds a light to understanding the regulation of myosin IX. We will further define the regulatory mechanism of myosin IX at the molecular and the cellular levels. In order to achieve the goal, we plan to use recombinant DNA technology as a means of production of engineered myosin IX and its mutants. The purified engineered myosin IX will be subjected to functional analysis by various biophysical, biochemical and electron microscopy techniques with particular emphasis on a single molecule assay system. The proposed project will clarify the function and regulation of myosin IX, a unique member of myosin super family, thus provide a new insight into the mechanism of actin based motility.